Despite the fact that animal-based neurotoxicology models have relatively low sensitivity, and are burdened by high workload, cost and animal ethics, they have been the mainstay of evaluating neurotoxicology. However, toxicology today is looking for alternatives as it faces enormous challenges to the use of animals. On one hand there is enormous societal pressure to reduce the use of animals, and on the other hand the demand for testing is increasing. More than 30,000 chemicals are estimated in use without adequate toxicological information in the USA and Europe, and the task of testing thousands of chemicals systematically with classical animal assays likely exceeds our present capabilities. The recent advance in pluripotent stem cell (PSC)-based technology and the ability to generate truly large numbers of allelically diverse cells and use uniform methods of differentiation into al the major types of cells in the nervous system offer a potential new tool for improved understanding of chemically induced toxicity. This is especially useful for developmental neurotoxicity, because neural cells differentiate early during development and this process is relatively easily recapitulated in vitro via rosette formation and isolation of neural stem cells (NSC), which can subsequently be differentiated into neurons and glia. We have in a Phase I grant provided proof-of-concept for iPSC-based neurotoxicity assays. However several issues need to be addressed before such assays can be used routinely to test neurotoxicity. For example, there is a lack of neural reporters in referenced lines of both genders, which will be invaluable for further assay development and refinement. There is also a lack of datasets that can serve as baseline for toxicity assays, and a lack of reference response to a reference set of compounds that can be used to calibrate the response of future lines and compare with the rodent data. The objective of this Phase II application is to develop a neurotoxicity tool kit that addresses these issues and to commercialize it.